Histopaque solution

The second and third portions of the fresh and 24 h blood samples were used to isolate PBMCs. Separation of PBMCs from blood samples was accomplished by gently layering 1.5 mL blood over 2 mL Histopaque® solution (Sigma-Aldrich, USA) and centrifuging at 1500 rpm for 30 min. The white band of mononuclear cells was harvested and washed 3 times with RPMI 1640 culture medium by centrifugation at 3000 rpm for 5 min. PBMCs were suspended in RPMI 1640 medium (containing 25 mM HEPES, 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, penicillin [100 U/mL], and streptomycin [100 g/mL]) and then adjusted to a concentration of 2×10⁶ cells/mL. Then, 200 μL suspended PBMCs were seeded into a 96-well plate and incubated at 41°C in a humidified atmosphere containing 5% CO₂ for 5 consecutive days.

Whole blood (2 mL), collected from EDTA blood collection tubes (Becton Dickinson, Franklin Lakes, NJ, USA) at 0, 2, and 4 h, was layered over 2 mL of Histopaque solution (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 30 min at 400× g at room temperature. PBMCs were removed from the interface, washed twice with phosphate buffered saline (PBS), and RNA was extracted (Purelink RNA Mini Kit, Life Technologies, Waltham, MA, USA). DNA contamination was eliminated by DNase digestion from the cell lysate prior to RNA isolation. RNA concentration was measured using a NanoDrop (ND-1000 Spectrophotometer, Thermo Scientific, Waltham, MA, USA). RNA was reverse transcribed using a High Capacity RNA-to-cDNA Kit (Life Technologies, Waltham, MA, USA) as per the manufacturer’s instructions.

This study was carried out with the approval of the Institutional Review Board (Helsinki Committee) and with the consent of the dementia patients or their guardians. The physicians diagnosed the cohort as controls, AD patients, and DLB patients using the classical methods, which are based on the evaluation of the patients’ medical histories; physical and laboratory imaging examinations such as MRI, CT, and PET; and the personal experience of the physicians who treated these patients for a long period of time. Blood samples were collected and analyzed to fulfill the objectives of the current study. WBC and plasma components were separated from whole blood samples (2 to 3 mL) within $<3\mathrm{h}$ after collection. The Hudson and Poplack method was followed to accomplish the separation process.⁴⁵ Briefly, 3 mL of Histopaque solution (Sigma Chemical Co., St. Louis, Missouri) was added to the blood tube before being centrifuged at 300 g for 30 min at 23°C. After centrifuging, the WBC, which appears as a layer located at the middle of the tube, was isolated and washed with phosphate-buffered saline by centrifugation at $300g$ for 10 min at 23°C. One microliter of WBC samples and the same amounts of plasma samples were mounted as separated drops on zinc selenide crystal, which is transparent to IR radiation. The samples were dried under laminar flow for about 15 min before measuring.